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PubMed2006

Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila.

Salt-sensitive glycophytes and salt-tolerant halophytes employ common mechanisms to cope with salinity, and it is hypothesized that differences in salt tolerance arise because of changes in the regulation of a basic set of salt tolerance genes. We explored the expression of genes involved in two key salt tolerance mechanisms in Arabidopsis thaliana and the halophytic A. thaliana relative model system (ARMS), Thellungiella halophila. Salt overly sensitive 1 (SOS1) is a plasma membrane Na+/H+ antiporter that retrieves and loads Na+ ions from and into the xylem. Shoot SOS1 transcript was more strongly induced by salt in T. halophila while root SOS1 was constitutively higher in unstressed T. halophila. This is consistent with a lower salt-induced rise in T. halophila xylem sap Na+ concentration than in A. thaliana. Thellungiella halophila contained higher unstressed levels of the compatible osmolyte proline than A. thaliana, while under salt stress, T. halophila accumulated more proline mainly in shoots. Expression of the A. thaliana ortholog of proline dehydrogenase (PDH), involved in proline catabolism, was undetectable in T. halophila shoots. The PDH enzyme activity was lower and T. halophila seedlings were hypersensitive to exogenous proline, indicating repression of proline catabolism in T. halophila. Our results suggest that differential gene expression between glycophytes and halophytes contributes to the salt tolerance of halophytes.

Anthocyanins↗
PubMed1995

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein.

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.

Amino Acid Sequence↗
PubMed1986

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids. The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains. The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions. The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.

Chromosome Mapping↗
PubMed1989

Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.

The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.

Cloning, Molecular↗
PubMed1981

Plant prolyl hydroxylase recognizes poly(L-proline) II helix.

Substrate specificity of a prolyl hydroxylase from Vinca rosea suspension-cultured cells was studied using synthetic oligo(L-proline)s and their t-butyloxycarbonyl derivatives (Pron and Boc-Pron; n = 2-8) as peptidyl substrates. All peptides with a residual number of 5 or greater served as substrates in the enzyme reaction at 30 degrees C, after the preincubation of the enzyme and peptides at 0 degrees C prior to addition of cofactors and cosubstrate. Under the same conditions, the hydroxylation of Pro5 reached a plateau within 10 min, but that of Boc-Pro8 and poly(L-proline) increased linearly up to 40 min. If the preincubation temperature was raised to 30 degrees C, only Pro5 among the peptides was unable to serve as a substrate. The optimum temperature of the enzyme was 30 degrees C toward Boc-Pro8 and poly(L-proline) but it decreased to 15 degrees C using Pro5. These data suggest that the enzyme can bind Pro5 only at low temperature. Poly(L-proline) and Boc-Pron (n greater than or equal to 5) in aqueous solution are known to have a left-handed helical structure (poly(L-proline) II helix). Moreover, Pro5 was indicated as forming this helix below 10 degrees C. Accordingly, the enzyme recognizes the poly(L-proline) II helix, that is, the secondary structure of a substrate rather than the primary structure.

Circular Dichroism↗
PubMed1983

Schistosomiasis: proline production and release by ova.

The production and release of proline was measured in cleaned ova of Schistosoma mansoni. Proline was found to be released at approximately 76 mumoles/100 cc of ova water/hr. This high rate of proline production was found to correlate with extremely active proline synthetic enzymes in the ova. Ornithine-delta-transaminase, which converts ornithine to delta 1-pyrroline-5-carboxylic acid, was found to be twice that of adult schistosomes and over seven times that of rat liver. delta 1-pyrroline-5-carboxylic acid reductase, which converts delta 1-pyrroline-5-carboxylic acid to proline, is also more active than the adult isoenzyme and 35 times more active than the rat liver isoenzyme. These data suggest that proline emanating from the ova may play a role in stimulating fibrosis in the granulomata of schistosomiasis.

Animals↗
PubMed2006

Impact of cis-proline analogs on peptide conformation.

The beta-turn is a common motif in both proteins and peptides and often a recognition site in protein interactions. A beta-turn of four sequential residues reverses the direction of the peptide chain and is classified by the phi and psi backbone torsional angles of residues i + 1 and i + 2. The type VI turn usually contains a proline with a cis-amide bond at residue i + 2. Cis-proline analogs that constrain the peptide to adopt a type VI turn led to peptidomimetics with enhanced activity or metabolic stability. To compare the impact of different analogs on amide cis-trans isomerism and peptide conformation, the conformational preference for the cis-amide bond and the type VI turn was investigated at the MP2/6-31+G** level of theory in water (polarizable continuum water model). Analogs stabilize the cis-amide conformations through different mechanisms: (1) 5-alkylproline, with bulky hydrocarbon substituent on the C(delta) of proline, increases the cis-amide population through steric hindrance between the alkyl substituent and the N-terminal residues; (2) oxaproline or thioproline, the oxazolidine- or thiazolidine-derived proline analog, favors interactions between the dipole of the heterocyclic ring and the preceding carbonyl oxygen; and (3) azaproline, containing a nitrogen atom in place of the C(alpha) of proline, prefers the cis-amide bond by lone-pair repulsion between the alpha-nitrogen and the preceding carbonyl oxygen. Preference for the cis conformation was augmented by combining different modifications within a single proline. Azaproline and its derivatives are most effective in stabilizing cis-amide bonds without introducing additional steric bulk to compromise receptor interactions.

Binding Sites↗
PubMed1980

L-proline as a glutamate antagonist at a crustacean neuromuscular junction.

The fast as well as the slow contractions of the adductor muscle in the claw of Procambarus clarkii are inhibited by L-proline. This inhibition is dose dependent and decreases with increasing frequency of stimulation of the "slow" fiber. Contractions caused by perfusing the adductor muscle with L-glutamate solutions are also inhibited by L-proline. The inhibiting potency of L-proline is small; the effective concentration of this amino acid is 50--100 times that of the L-glutamate applied. It was postulated that the inhibitory effect of L-proline is based on competition for excitatory receptor sites of L-glutamate, which causes depolarization and contraction, and L-proline, which lacks these actions. Theoretical considerations suggested a linear relationship between the stimulating L-glutamate and the just-inhibiting L-proline concentrations. Experimental evidence supported this model.

Animals↗
PubMed1995

Proline in a transmembrane helix compensates for cavities in the photosynthetic reaction center.

A site-specific double mutant, in which the large aromatic residues M208Tyr and L181Phe in the interior of the photosynthetic reaction center (RC) complex were replaced by smaller threonine residues, showed a dramatic reduction in the number of assembled complexes and was incapable of photosynthetic growth. The cavity created by the smaller side-chains was thought to interfere with the stability and/or assembly of the complex. Phenotypic revertants were recovered in which a spontaneous second-site mutation restored photocompetence in the presence of the original site-specific mutations. In these strains, an Ala-->Pro substitution in a neighboring transmembrane helix (at M271) resulted in an increased yield of RC complexes. To test the hypothesis that the original phenotype was due to a cavity, other mutants were constructed that created similar-sized voids at other positions in the membrane-spanning interior. These substitutions caused the same phenotype. Coupling of the above proline substitution to these new cavity mutants also resulted in photocompetent strains that carry increased levels of RC complexes. Therefore, the proline substitution at M271 serves as a global suppressor of the phenotype caused by these internal cavities. The proline substitution slightly increases the thermal stability of the complex at higher temperatures, but the mutant and suppressor strains have about the same stability at the optimal culture temperature, where both are less stable than the wild-type strain. Therefore, the proline substitution may suppress the non-photosynthetic phenotype of cavity mutants by facilitating folding of the nascent polypeptides as they assemble with cofactors to form the transmembranar RC complex. The proline replacement occurs at a pre-existing kink in a transmembrane helix where it can be accommodated without introducing a strain in the structure. The function of proline residues in transmembrane helices might be to promote folding and/or assembly in general.

Cell Membrane↗
PubMed1986

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12.

Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product). Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.

Amino Acid Transport Systems, Neutral↗
PubMed1984

High-frequency induction by 5-azacytidine of proline independence in CHO-K1 cells.

Proline independence in CHO-K1 Chinese hamster cells has in previous studies been characterized as an auxotrophic gene mutation. In the absence of direct proof, an alternative model must be considered, based on suppression of proline synthesis by DNA methylation changes at one or more loci concerned. This concept receives strong support from the present study, in which we show that treatment of CHO-K1 cells with 5-azacytidine induces a 10(5)-10(6) increase in background conversion to the proline-independent state. Revertants thus obtained, as well as those arising spontaneously or after treatment with ethyl methane sulfonate, are stable phenotypically in the presence or absence of proline. Proline independence in all variants examined was correlated with increased activity of pyrroline-5-carboxylate synthase. Four of five variants induced with 5-azacytidine showed simultaneous increases in activity of ornithine aminotransferase as well. Our data suggest that epigenetic, rather than genetic changes, underlie the transitions between proline dependence and independence in CHO-K1 cells.

Animals↗
PubMed1989

Uptake of lysine and proline via separate alpha-neutral amino acid transport pathways in Mytilus gill brush border membranes.

Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel, Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na+ and alpha-neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar alpha-neutral amino acids and cationic amino acids, but was not affected by beta-neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with a Jmax of 550 pmol/mg-min and a Kt of 5 microM. The AK pathway did not have a strict requirement for Na+, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li+ and K+, as well as Na+. Harmaline inhibited the transport of lysine in solutions containing either Na+ or K+. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar alpha-neutral amino acids, proline, and alpha-(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with a Jmax of 180 pmol/mg-min and Kt of 4 microM. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na+. Na+-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na+ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.

Alanine↗
PubMed1985

Na+ (Li+)-proline cotransport in Escherichia coli.

Na+ and Li+ were found to stimulate the transport of L-proline by cells of Escherichia coli induced for proline utilization. The gene product of the put P gene is involved in the expression of this transport activity since the put P+ strains CSH 4 and WG 148 show activity and the put P- strain RM 2 fails to show this cation coupled transport. The addition of proline was found to stimulate the uptake of Li+ and of Na+. Attempts to demonstrate proline stimulated H+ uptake were unsuccessful. It is concluded that the proline carrier (coded by the put P gene) is responsible for Na+ (or Li+)-proline cotransport.

Biological Transport, Active↗
PubMed1979

Changes in proline synthetic and degradative enzymes during matrix-induced cartilage and bone formation.

Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3 days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.

Animals↗
PubMed1987

On the specificity of prostaglandin and steroid induced inhibition of 3H-proline incorporation in the human follicular wall.

Experiments were carried out to elucidate whether the previously reported inhibitory effect of prostaglandin (PG) E2 on 3H-proline incorporation into total protein of specimens from the isolated preovulatory human follicular wall could be exerted also by other prostanoids. Moreover, investigations were designed to explore if the previously documented inhibitory effect of steroids on 3H-proline incorporation could be mediated by endogenously formed PGs. Tissue specimens from the apical wall of follicles were incubated in the presence of PGF2 alpha, 6-keto-PGF1 alpha, progesterone (P) or 17 beta-estradiol (E2). The steroids were added alone or in combination with the PG synthetase blockers, indomethacin or 5, 8, 11, 14-eicosatetraynoic acid. Following incubation with 3H-proline for 2-4 h, the incorporated radioactivity was determined. PGF2 alpha and 6-keto-PGF1 alpha had no effect on 3H-proline incorporation. Both P and E2 induced a significant decrease of 3H-proline incorporation in preovulatory follicles, whereas similar effects in unripe follicles were statistically significant only for P. The steroid effects were not influenced when blockers of PG synthesis were added concomitantly. It is concluded that among the "classical" PGs PGE2 has a unique effect on collagen metabolism in the human follicular wall. E2 and especially P have a similar inhibitory effect on collagen formation as reflected by measurements of 3H-proline incorporation, an effect which does not appear to be primarily mediated by endogenous PGs.

Collagen↗
PubMed1998

Voltage sensitivity and conformational change of isolated S4L45 fragments from sodium channels are tuned to proline.

Peptide fragments reproducing the sequences of S4 segments extended with L45 linkers from the four homologous domains of the electric eel sodium channel were chemically synthesized and purified to allow circular dichroism studies in various solvents and conductance assays in planar lipid bilayers. Repeats III (with proline) and IV (lacking proline) present the lowest and highest helicities, respectively. The conformational transition (from helix to beta-strand) shown to occur on an increase of solvent dielectric constant is broader with repeat III. Analytical ultracentrifugation (interference fringe pattern) is consistent with a monodispersion of the peptide. In macroscopic conductance experiments, the proline containing peptides (repeats I, II and especially III) display higher voltage-sensitivities than repeat IV. The apparent and averaged number of monomers per intramembrane conducting aggregate is 4-5. The influence of proline is confirmed in similar experiments carried out on homologous S4 segments of repeat IV of the human skeletal muscle sodium channel comparing the wild type and an analogue where the fourth arginine was substituted with a proline. Thus, both conformational switching and voltage-sensitivity appear correlated to the presence and position of a single proline residue. Since voltage sensors are likely to experience different polarity environments in the channel open and closed states, our results suggest an alternative gating mechanism, i.e. a voltage-driven conformational change of S4L45s. The data also implies a plausible functional asymmetry, namely a "three- or four-stroke" activation sequentially involving the four domains of the sodium channel.

Amino Acid Sequence↗
PubMed2006

Free radical scavenging potential of L-proline: evidence from in vitro assays.

An assessment of the potential of proline to scavenge free radicals was made in a couple of in vitro assay systems, namely graft co-polymerization and autooxidation of pyrogallol. Both these assays are essentially dependent upon free radical mechanisms. Graft co-polymerization involved a ceric (Ce(4+)) ion- or gamma-radiation-induced grafting of methyl acrylate (MA) onto a cellulose backbone. The degree of grafting, measured gravimetrically, was taken as a measure of free radical generation. The gamma-radiation-dependent grafting was far greater than that due to Ce(4+) ions. Inclusion of proline in the assay, irrespective of the initiator used, led to suppression of grafting in a concentration-dependent manner indicating the ability of proline to scavenge free radicals. The gamma-radiation-dependent grafting was also suppressed by hydroquinone and glutathione but not by ascorbate, glycine and spermine. In contrast to graft co-polymerization, proline did not inhibit the autooxidation of pyrogallol, a reaction involving superoxide radical generation. A subset of data constitutes an evidence for the ability of proline to scavenge free radicals in vitro. It is implied by extension that free proline, known to accumulate in plant tissues during abiotic stresses, would contribute to scavenging of surplus free radicals produced under a variety of abiotic stresses.

Acrylates↗
PubMed1982

L-Proline transport in Saccharomyces cerevisiae.

Transport of L-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a KT of 31 microM and Jmax of 40 nmol . s-1 . (g dry wt.)-1, the other with KT greater than 2.5 mM and Jmax of 150-165 nmol . s-1 . (g dry wt.)-1. The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) L-proline and its analogs L-azetidine-2-carboxylic acid, sarcosine, D-proline and 3,4-dehydro-DL-proline, and (ii) L-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8-5.9 and, in an Arrhenius plot, exhibits two inflection points at 15 degrees C and 20-21 degrees C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50-60% of accumulated L-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, the pH between 3 and 7.3, as well as to the presence of 10-100 mM unlabeled L-proline in the outside medium. Its rate and extent are increased by 1% D-glucose and by 10 micrograms nystatin per ml.

2,4-Dinitrophenol↗